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Definition and characterization of an artificial En/Spm-based transposon tagging system in transgenic tobacco

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Abstract

A transposon tagging system for heterologous hosts, based on the maize En/Spm transposable element, was developed in transgenic tobacco. In this system, the two En-encoded trans-acting factors necessary for excision are expressed by fusing their cDNAs to the CaMV 35S promoter. The dSpm receptor component is inserted in the 5′-untranslated leader of the bar gene. Germinal revertants can therefore be selected by seed germination on L-PPT-containing medium or by spraying seedlings with the herbicide Basta. Using this bar-based excision reporter construct, an average frequency of germinal excision of 10.1% was estimated for dSpm-S, an En/Spm native internal deletion derivative. Insertion of En-foreign sequences in a receptor, such as a DHFR selectable marker gene in dSpm-DHFR, does not abolish its capacity to transpose. However, dSpm-DHFR has a lower frequency of somatic and germinal excision than dSpm-S. Revertants carrying a transposed dSpm-DHFR element can be selected with methotrexate. Germinal excision is frequently associated with reinsertion but, as in maize, dSpm has a tendency to integrate at chromosomal locations linked to the donor site. Concerning the timing of excision, independent germinal transpositions are often found within a single seed capsule. All activity parameters analysed suggest that transposon tagging with this system in heterologous hosts should be feasible.

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  1. Abteilung Molekulare Pflanzengenetik, Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, 50829, Köln 30, Germany

    Guillermo H. Cardon, Monika Frey, Heinz Saedler & Alfons Gierl

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  1. Guillermo H. Cardon
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  2. Monika Frey
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  3. Heinz Saedler
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  4. Alfons Gierl
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Cardon, G.H., Frey, M., Saedler, H. et al. Definition and characterization of an artificial En/Spm-based transposon tagging system in transgenic tobacco. Plant Mol Biol 23, 157–178 (1993). https://doi.org/10.1007/BF00021428

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